Attenuated live rubella virus vaccine and method of production

ABSTRACT

PRODUCTION OF AN ANTIGENICALLY ACTIVE, ATTENUATED, LIVE RUBELLA VIRUS VACCINE BY INTRODUCING A VIRULENT LIVE RUBELLA VIRUS INTO A MONKEY KIDNEY TISSUE CELL CULTURE, INCUBATING THE TISSUE CELL CULTURE AT A TEMPERATURE COMPATIBLE WITH GROWTH OF THE TISSUE AND THE VIRUS, HARVESTING A PORTION OF THE VIRUS SO-PRODUCED AND REINTRODUCING SAID HARVESTED VIRUS INTO FRESH CULTURES, AND REPEATING THE TISSUE CULTURE PASSAGES OF THE VIRUS SERIALLY FOR A SUFFICIENT NUMBER OF PASSAGES TO PRODUCE THE ANTIGENICALLY ACTIVE, NONCOMMUNICABLE, LIVE RUBELLA VIRUS AND THEN FORMING A VACCINE THEREFROM.

March 21, 1972 R, JR E'I'AL 3,651,212

ATTENUATED LIVE RUBELLA VIRUS VACCINE AND METHOD OF PRQDUCTION FiledDec. 20, 1966 2 Shoets Sheetl1 4.0 F/aj GMK PASSAGE LEVEL RUBELLA -V\RUS'HTER \ND\CA'\'ED H BY cYToDATmc H 4 EFFECT. T

(Loa -K0 M O DAYS AFTER \NOCULA'\'\ON FIG. 2 3o L0 CUMULATWE NUMBER \mTHANT\BODY O :1 NUMBER TES ED BLOOD T SPECMENS 1 T E?) NUMBER WITH VlRUSTESTED FOR VHZUQ Q 1 1 T I o THROAT SwABs TESTED FOR '10 \mzus ox S \o\5 1o '15 '50 vg v z DAYS AFTER \NOCULAT\ON f/AQQYMMEYEQJK March 21,1972 MEYER, JR" EI'AL 3,651,212

ATTENUATED LIVE RUBELLA VIRUS VPICCINE AND METHOD OF PRODUCTION FiledDec. 20. 1966 2 Sheets- Sheet 2 31. i we ANUBODY 2 UTEIZ H i I14 1 1* Ii i o o g .2 8 2 DAYS AFTER \NOCULA'HON .-.J@ @@@@@@@@@@@ga@ CUMULAT \VENUMBER A W \T H AN'HBQDY [:1 NUMBER TESTED BLQQD spacmeus l NUMBE rmvuzus TESTED FOR 4 l R w THRO AT SWABS TESTED FOR \IUZUS DAY ONSET RA$H///I/?/?YM Meme PAUL 0. PAR/(MAN 5y s/acdSi 011x480 ATTORNEK? UnitedStates Patent Oflice 3,651,212 Patented Mar. 21, 1972 3,651,212A'ITENUATED LIVE RUBELLA VIRUS VACCINE AND METHOD OF PRODUCTION Harry M.Meyer, Jr., Silver Spring, and Paul D. Parkman, Kensington, Md.,assignors to the United States of America as represented by theSecretary of the Department of Health, Education, and Welfare Filer Dec.20, 1966, Ser. No. 603,239 Int. Cl. C12k 5/00, 7/00 US. Cl. 424-89 8Claims ABSTRACT OF THE DISCLOSURE Production of an antigenically active,attenuated, live rubella virus vaccine by introducing a virulent liverubella virus into a monkey kidney tissue cell culture, incubating thetissue cell culture at a temperature compatible with growth of thetissue and the virus, harvesting a portion of the virus so-produced andreintroducing said harvested virus into fresh cultures, and repeatingthe tissue culture passages of the virus serially for a suflicientnumber of passages to produce the antigenically active, noncommunicable,live rubella virus and then forming a vaccine therefrom.

This invention is concerned with the production of an antigenicallyactive, attenuated, live rubella virus vaccine.

The rubella virus infection, commonly known as German measles, isprimarily a disease of children and young adults and is clinicallycharacterized by sore throat, coryza, headache, malaise, myalgia,posterior cervical lymphadenitis and a pale pink macular rash. Accurateinformation on the incidence of the disease is not available becausestudy has been limited by the virus restrictive host range (humans andmonkeys) and by the difliculty in diagnosing so mild a disease. -It hasbeen established, however, that occurrence of rubella infection isworld-wide.

Rubella infection is highly contagious. The virus is probablycommunicated via the respiratory route by close personal contact and isusually detectable both in the blood and in nasopharyngeal washings.

The, usual course of the disease leads to prompt and complete recoverythough relapse occurs in 5-8 percent of the cases. Secondary bacterialinfections are rare as are other complications which include arthralgia,neuritis, gingivitis, thrombocytopenic purpura and heart block.Meningoencephalitis occurs in one of every 6,000 cases and is 20 percentfatal. A patient who has contracted and has recovered from a rubellainfection usually enjoys lasting immunity from subsequent attacks.

The most dismal aspect of this disease is that it is the only viralinfection proven to be associated with fetal abnormalities in caseswhere a pregnant woman contracts the disease in her first trimester ofpregnancy. Though it is not true, by any means, that abnormalitiesalways result in the offspring of a first-trimester infected pregnantwoman (she has a 90 percent chance of bearing a normal baby), whenabnormalities do occur they are he quently so severe as to lead tointrauterine fetal death, stillbirth, or delivery of a viable infantwith tragic defects such as microencephaly, dental hypoplasia, blindnessdue to cataract formation, deafness due possibly to agenesis of theOrgan of Corti and acyanotic cardiovascular disease such as patentductus arteriosus and intraventriculas septal defects.

Since rubella frequently infects young adults, there is a real danger ofits occurrence during pregnancy. Motivated by the severity of thisproblem, a great deal of work has been done in attempting to conferimmunity on susceptible individuals. Early approaches toward theproduction of a killed-virus vaccine were unsuccessful. Further, varioustissue culture propagated live rubella viruses produced prior to theinstant development were also unsuccessful. Previously tested live-virusvaccines have not been able to stimulate the immuno logic mechanism ofan inoculated individual without producing the ordinary side effects ofclinical rubella including the appearance of the rash and the spread ofthe disease to uninoculated persons.

Thus, the basic object of this invention is the production of anattenuated live rubella virus vaccine which retains its antigenicproperties and is capable of providing immunity to reinfection to aninoculated, otherwise susceptible, individual without producingundesirable reactions ordinarily associated with contagious rubella.Further, a primary object of this invention is to provide a method forattenuating live rubella virus and for prepai ing a vaccine therefrom inan efiicient and reproducible manner.

A corollary to the aforementioned objectives of this invention is thedevelopment of laboratory marker techniques useful in detectingmodification of a rubella virus strain before its clinical trial in manwhereby attenuation of the virus will be signalled by the presence ofsuch changes in virus behavior.

Other objects will in part be obvious and in part be pointed out as thedescription of the invention proceeds and as shown in the accompanyingdrawings wherein:

FIG. 1 is a graph showing the effect of low and high passage levelrubella virus on cytopathic changes in RKlg rabbit kidney cells, one ofthe marker techniques utilized in developing the vaccine of the instantinvention;

FIG. 2 is a graphic illustration summarizing the virologic eventsfollowing the inoculation of a total of 34 susceptible children duringthe clinical trials of the vaccine of this invention;

FIG. 3 shows the level and duration of neutralizing antibodies in thechildren inoculated during the clinical studies of the vaccine producedaccording to the instant inventive concepts; and

FIG. 4 shows comparative virologic events in an outbreak of naturalrubella.

In order to distinguish an attenuated rubella virus produced accordingto this invention from an unmodified, natural rubella virus, the latterwill be referred to herein and in the appended claims as virulent.Further, persons identified herein as susceptible to rubella are thosehaving no detectable neutralizing antibody of rubella.

Having now provided a definition for certain key terms employed in thisdescription, reference will now be made to the basic laboratorytechniques utilized in the production of an attenuated live rubellavirus vaccine according to this invention.

Virulent rubella virus strains, both M-33 and ML, were recovered fromthroat washings of patients with typical clinical rubella. Both viruseswere subjected to several levels of tissue-culture passage, the M-33strain being passed over times and the ML strain being passed 70 times.The two rubella virus strains were serially subcultured in primaryAfrican green monkey kidney cells (GMK), supernatant fluids and cellsfrom the rubellainfected cultures being passed at intervals of seven tonineteen days. Although reference will be made herein to both the M-33and the ML strains, primary emphasis will be directed to the use of theM-33 strain since the original development utilized this material.However, various test results will be reported hereinafter evidencingthe production of an attenuated virus from the ML strain as well as fromthe M-33 strain.

The techniques utilized for producing and safely testing a live virusvaccine from the M-33 rubella strain we're as follows VIRUS (a) Seedstrain.The virulent M-33 virus strain was recovered from an Army recruitwith rubella at Fort Dix, New Jersey. This recruit had a mild illnesslasting three days with generalized rash and posterior cervicallymphadenopathy. The patieint had a low-grade fever for one day.

Throat swabs were negative for :phemolytic streptococci. Throat washingsyielded an interfering agent in GMK. In subsequent studies, this viruswas shown to be the etiologic agent of rubella.

(b) Passage hisIry.-The rubella virus was recovered in GMK andsubsequently, serially passaged in this cell type 77 times. Ordinarily,in these passages, 0.2 ml. of undiluted supernatant fluids and scrapedcells were subcultured at 7 to day intervals in cultures maintained at35 to 36 C. in stationary racks. Exceptions to this procedure were madeat the 5th-6th and 69th-73rd and 75th passage levels. The 5th and 6thwere incubated at 32 C. in roller drums and the 69th-73rd and 75thpassage levels were made as limiting infectiousdilutions forpurification.

The high passage modified virus which forms the basis of most of theclinical trials reported hereinafter has been designated HPV-77.

(c) Tests on seed.The seed virus pool used for the inoculation of tissuecultures for vaccine production was shown to be free of demonstrableextraneous, viable agents by tests in tissue cultures to excludecytopathic viruses, and in appropriate media to exclude bacteria, fungiand pleuropneumonia-like organisms (PPLO).

PREPARATION OF LIVE RUBELLA VIRUS VACCINE (a) Facilities andpersom'nel.-All processing steps up to and including filling into finalcontainers of the rubella virus seed pool and vaccine were performed ina cubicle which formed an independent unit in a room. Prior to use forthis purpose, the area was decontaminated and the cubicle was not usedfor any other purpose during preparation of the vaccine.

On days when the vaccine was being processed, personnel avoided workwith other infectious agents. Face masks and gowns were worn in thevaccine processing unit.

(b) Tissue culture production, maintenance and inoculatiom-Tissuecultures for vaccine production were prepared from kidneys of Africangreen monkeys, guarantined and tuberculin tested. Culture vessels (32oz. prescription bottles) were seeded with trypsinized suspensions ofgrivet monkey kidney containing 300,000 viable cells per ml. in Hankslactalbumin hydrolysate medium with 5 percent calf serum (inactivated at56 C. for 30 minutes prior to use). Three days after seeding, thevessels were changed with 35 ml. Eagles minimum essential medium (Ml-3M)containing 5 percent fetal bovine serum and neomycin, 25 mg./ml. Whencell moonlayers were confluent (6 days after seeding) the vessels werere-fed with maintenance medium consisting of MEM, 1 percent fetal bovineserum and the same concentrations of neomycin.

After observation for three days, the supernatant fluids were drawn offand pooled for the safety tests applied to pre-inoculation culturefluids (see below). Each vessel was then re-fed with 35 ml. ofmaintenance medium. Twelve of the cultures were inoculated with 1 ml.each of a 1:10 dilution of the primary seed virus and the remaining sixwere held as uninoculated cell controls. All cultures were incubated at35 C.

(c) Vaccine harvest.-On the 7th day after inoculation, all cultures werewashed three times with Hanks balanced salt solution (BS8) to reduce theserum protein concentration to less than 1 part per million, and re-fedwith medium 199 (M-199) 3 with 25 mg./ml. neomycin.

1 MEM composition as follows: GmJliter l-arginine HCl 0.126 l-cystineQ02 l-glutamine 0292 l-histidine 0,031 l-isoleuclne 0.052 l-leucine0.052 l-lysine HCl 0.073 l-methionine 0.015 l-phenylalanine 0.032l-threonine 0.048 ltryptophan 0.01 l-tyrosine 0.036 l-valine 0.046

MgJliter Choline chloride 1.0 Folic acid 1.0 Nicotlnamide 1.0 Capantothenate 1.0 Pyridoxal HCl 1.0 Thiamine HCl 1.0 iltboiiisvlln nos 0Gnu/liter Nacl 6.8 KCl 0.4 NaHaPOrHzO 0.15 NaHCOa 2.2 CaCls 0.2MgClsfiHaO 0.2 Dextrose 1.0 Phenol Red w.s. 0.005 BSS composition asfollows: Gm./liter NaCl 8.0 K01 0.4 MgSOflHaO 0.1 MgClaGHzO -1 CaCh 0.15NaaHPOelZHaO 0.152 KHL'POL 0.06 Dextrose 1.0 Phenol Red NaHOOa SterileDM Dist. H20 to 1,000 mi.

lid-199 composition as follows:

l-arginine i-histidine 2 l-lysine H01 007 dl-Tryptophate(ll-Phenylalanine 0- til-Methionine dl-Serine 0.05 dl-Threonine 0.06dl-Leucine 0.12 dl-Isoleucine 0.04 dl-Valine 0.05 dl-Glutamic acid 0.15dl-Aspartic acid 0.06 dl-Alanine 0.05 1- roline 0.04 1- ydroxyproline0.01 Glycine 0.05 1-glutamine 0.10 Na acetate 0.05 l-tyrosine 0.04l-cystine 0.02 Niacinamide 2.5 10- Niacin 25x10- Pyridoxine HCl 2.5)(10-Pyrodoxal H01 2.5 10- Thiamine HCl Riboflavin Ca paintothenate 1.0)(10i-Inositol 5.0)(10- p-Aminobenzoic acids 50x10" Choline chloride 5.0X1O-Biotin 1.0X10- Folic acid 1.0X10- Calciferol (vit. Dz) 1.0 10-Cholesterol 2.0)(10- Sorbitan mono-oleate (Tween 0.02 d-Alpha tocopherolacetate (vit. E) 1.0)(10- Vitamin K 1.0)(10- Adenine sulphate Xanthine'Hypoxanthine 3.0X10- Thymine 3.0 10- Uracil 3.0X10- Guanine 3.0X10-d-a-Desoxyribose 5.0 10- d-Ribose 5.0 10- Adenylic acid 2.0X10- Ferricnitrate (Fe(NOs)-9H=O) 1.0 10- Cysteine HCI 1.0X10- Glutathione 5.0)(10-Ascorbic acid 5.0)(10- Vitamin A 1.0X10- Ethyl alcohol (EtOH), ml. 0.201Adenosine triphosphate (ATP) 0.01 8.0

NaQl (biol. grade) KCl MgSOr'IHaO 0.2

One day later, and for a total of 7 days, harvests were collected at 24hour intervals and after each harvest, the cultures were re-fed withfresh M-199. Processing of viral vaccine fluids was under aspeticconditions. Samples from each harvest were removed for microbialsterility tests and infectivity titration, and the remainder of the poolwas frozen at -70 C. Supernatant fluids from uninoculated controlvessels were similarly harvested and stored. Aliquots of the harvests ofinoculated and control vessels collected from the 8th day to the th dayafter inoculation were separately pooled and safety tested. The rubellavirus pool (formed from aliquots of the individual virus harvests) wasthawed and clarified by centrifuge for minutes at 2000 r.p.m. Normalserum albumin (human) salt poor was added as a stabilizer to a finalconcentration of 2 percent. This final bulk material was dispensed intoindividual containers which were immediately frozen at 70 C. and labeledfor various tests.

With regard to the above preferred technique for producing an attenuatedlive rubella virus vaccine, it is to be understood that certain changesmay be made without departing from the scope of the instant invention.For example, although primary African green monkey kidney cell cultureswere utilized, other cultures which are capable of supporting growth ofthe virus may be substituted therefor including, but not necessarilylimited to, rhesus monkey kidney cell cultures, human embryo kidney cellcultures, rabbit kidney cell cultures, avian embryo cell cultures anddiploid cell cultures. Also, although incubations were carried out atabout C., it should be understood that the temperature may vary with thetissue culture system, and the basic requirement is that the cultures beincubated at a temperature compatible with growth of the tissue and thevirus. Similarly, the harvesting intervals should provide adequateopportunity for growth of the virus and the various nutrients should besuitable for sustaining the virus growth. Various purifying andstabilizing techniques Well known in the art may be utilized in theproduction techniques and concentration of the virus for vaccinepreparation may be accomplished in any desired manner.

TESTS FOR SAFETY The live rubella virus vaccine was tested for safety inaccordance with the Public Health Service Regulations Title 42, Part 73,for live, oral poliovirus and live, attenuated measles virus vaccines.

(a) Tests performed on pres-inoculation culture supernatants anduninoculated control tissue culture fluids.- Supernatant fluids from thepre-inoculation harvest and the pooled control fluid harvests wereinoculated into primary grivet and rhesus monkey kidney, human embryokidney and rabbit kidney cell cultures. A total of 20 ml. of fluid wasinoculated into 20 tubes of each tissue culture cell type. Thesecultures were observed for cytopathic effect, tested for hemadsorptionand the human and simian cultures challenged with echovirus type 11 forevidence of interfering agents. All safety tests were observed for aminimum of days including initiation of the cell cultures used forvaccine production. To achieve this observation period, subpassage ofthe pre-inoculation fluid harvest was maintained for 30 days and thepooled control fluid harvests for 17 days.

No cytopathic, hemadsorbing or interfering agents were detected in anyof these cell cultures and the tests were judged to be satisfactory.

(b) Tests performed on vaccine prior to clarification.- Aliquots fromeach of the seven daily harvests were thawed, pooled, cultured formicrobial sterility and titrated for rubella virus infectivity on theday of safety testing.

(i) Animal inoculation Rabbits.-15 healthy, 1500-2500 gm. rabbits wereeach inoculated with 0.1 ml. of vaccine intraderrnally, 111 10 sites andsubcutaneously with 4.5 ml. in each of 2 sites. All of the rabbitsremained healthy throughout the 21 day test period. No lesions wereobserved at the sites of inoculation and there was no evidence offl-virus or any other viral infection.

Adult mice-Thirty adult mice (15 to 20 gm.) were inoculatedintraperitoneally with 0.5 ml. and intracerebrally with 0.03 ml. ofvaccine. All survived the 21 day test period. None of the mice showedevidence of lymphocytic choriomeningitis virus or other virus infection.

Suckling mice.-Each of 29 suckling mice less than 24 hours old wereinoculated intracerebrally with 0.01 ml. and intraperitoneally with 0.1ml. of the vaccine. The litters were observed daily for 14 days. All 29mice survived the test. A blind passage of pooled, emulsified tissues(minus skin and viscera) from the surviving animals was made inadditional suckling mice, by intracerebral and intraperitoneal routesand these animals were observed for an additional 14 days. Noneevidenced symptoms suggestive of virus infection.

Guinea pigsFEach of 10 guinea pigs (350-450 gm.) was inoculatedintracerebrally with 0.1 ml. and intraperitoneally with 5.0 ml. of thevaccine. The animals were observed 42 days. There was no evidence of atransmissible agent attributable to the vaccine.

(ii) Tissue culture inoculation Primary grivet and rhesus monkey kidney,human embryo kidney and rabbin kidney culture vessels (32 oz.)containing 40 ml. of maintenance medium were each inoculated with anequal volume of vaccine and observed for 14 days for evidence oftransmissible agents. The total volume of vaccine inoculated into eachcell type was:

Ml. Primary grivet kidney 280 Primary rhesus kidney 320 Human embryokidney 390 Primary rabbit kidney 600 No cytopathic agents were detectedin any of the cultures.

(iii) Tests for pleuropneutnonia-like organisms (PPLO) Tests for PPLOwere performed in accordance with regulations for live, attenuatedmeasles virus vaccine (PHS Regulations para. 73.152 ii). There was noevidence of PPLO.

(iv) Bacteriological tests The vaccine was cultured in thioglycollateand Sabourauds broth for evidence of contamination with bacterial andfungal agents. Each medium was inoculated with a total of 20 ml. of thevaccine pool. Thioglycollate broth cultures were incubated for 7 days at32 C.; Sabourauds broth at room temperature (21 to 25 C.) for 10 days.No evidence of bacterial or fungal growth was observed in thioglycollatebroth when examined on the 3rd and 7th days or in Sabourauds broth onthe 10th day. These tests are satisfactory.

Tests for Mycobacterium tuberculosis were performed on the sedimentproduced by centrifugation of 20 m1. of the bulk sample for 20 minutesat 2500 r.p.m. The sediment was resuspended in 0.5 ml. of supernatant.Four LoWenstein-Iensen agar slants were each inoculated with 1 loopfulof this concentrate; four agar slants with 5 percent human blood wereeach inoculated and streaked with 0.1 ml. Cultures were incubated at 35C. There was no evidence of bacterial growth.

(c) Tests performed on vaccine after clarification. (i) Tissue cultureinoculation of virus pool after neutralizing with specificantiserum.--Equal volumes of the virus pool clarified by centrifugationand neutralized by specific rubella virus antiserum (rabbit) diluted inHanks BSS containing 10 percent normal rabbit serum, were incubated atroom temperature for one hour. One ml. of the neutralized mixtures andvaccine incubated similarly with diluent alone were inoculated intoprimary grivet and rhesus 7 monkey kidney, human embryo kidney andrabbit kidney cultures grown in screw cap tubes. These cultures anduninoculated cell controls were observed for cytopathic agents for 14days. At the end of the test period, one half of each group of cultureswas challenged with echovirus type 11. The final results were fullysatisfactory.

(ii) Neurovirulence safety test in monkeys for neu-rotropic agents (withcortisone).--Tests for neurovirulence in cortisone-injected monkeys wereconducted in accordance with the PHS regulations (73.102e), alsoproducing fully satisfactory results.

(iii) Neurovirulence safety test of virus strain.-Studies for thedetection of neurovirulence in rubella-susceptible monkeys wereconducted. The clarified bulk virus was inoculated into 20 rhesusmonkeys via the intrathalamic (0.5 ml. bilaterally) and intracisternalroutes (0.25 ml.).

Ten animals were sacrificed 21 days after inoculation and their centralnervous system tissues examined for presence of histopathologicalabnormalities. The remaining 10 monkeys were each caged with an animalsimilarly inoculated with uninfected control fluids from the same lot oftissue culture used for vaccine production. Five additional controlanimals (unnoculated) were caged adjacent to the inoculated animals.Detailed virologic studies were done on these additional animals andproduced fully satisfactory results.

FINAL CONTAINER TESTS (a) Potency tests.The vaccine was tested forpotency by titration of infectivity in primary grivet monkey kidney cellcultures. Individual vials of final container vaccine were thawed anddiluted serially in 0.5 log steps from 10 to 0.1 ml. of each dilutionwas inoculated into each of five tube cultures. Cultures were incubatedat 35 C. Medium was changed at 5 and again at .10 days when the cultureswere tested for evidence of interference wtih echovirus type 11.Interference endpoints were calculated by the Karber method and adjustedto the number of 50 percent interfering doses (lnD per 0.5 ml. The finalcontainer virus titer was '10- InD /0.5 ml.

(b) General safety tests.Two 350-450 'gm., Hartley strain guinea pigswere each inoculated intraperitoneally with 5 ml. of vaccine. Two adult,white mice were similarly inoculated, each with 0.5 ml. All animalssurvived a 7 day observation period without signs of illness.

(0) Sterility.The contents of 20 final containers were each inoculatedinto 500 ml. of thioglycollate broth; incubated at 32 C. and observed 7days for evidence of microbial growth.

The same volume of final container material was tested in Sabouraudsbroth. On completion of the observation period, fully satisfactoryresults were realized.

(d) Jdentity.Undiluted and l0 dilutions of final container vaccine weremixed with equal volumes of serial two-fold dilutions ofheat-inactivated rubella antiserum (rabbit) or normal rabbit serum.These serumvirus mixtures were incubated for one hour at 37 C. andinoculated into each of three grivet monkey kidney tube cultures.Appropriate controls were included. Neutralization of the virus in thevaccine constituted proof of its identity.

Thus, it will be seen that the resultant vaccine satisfied all of thelaboratory safety tests. The passage history set forth above, whilepreferred, is not to be considered limiting on the instant inventiveconcepts. Similar techniques are utilized in the production of a liverubella virus vaccine from the ML strain and similar results arerealized.

At intervals of 10 to 20 passages the above viruses were examined forindication of a change in their biologic characteristics. To precludethe necessity for clinical trials of vaccine which had not been properlyattenuated, the following in vitro marker techniques were utilized toevidence the presence of virus modification:

Cytopathic efiect (C'PE) in RK cellsfi-The first marker is theproduction of a cytopathic eifect with the virus in 'RK cells. Rubellaviruses do not produce extensive cell destruction in most tissue-culturesystems. Ordinarily, presence of rubella virus is detected by itscapacity to interfere with superinfection by a second virus, and thisproperty of interference has been used as a standard technique forquantitative titration of rubella-virus infectivity. However, rubellavirus will produce rapid and complete cytopathic changes in RK cellcultures, but only after 3 to 10 passages in homologous cells. In thissystem, infectivity can be expressed in terms of cytopathic change (TCDIt was, therefore, surprising that high GMK passage levels of rubellavirus produced degenerative changes in the heterologons RK cell system 5upon first passage. This unique characteristic of the high-passage virusprovided one of the laboratory markers.

Comparisons were made of the ability of several rubella-virus passagelevels to produce CPE in RK cells incubated at 35 C. An illustrativetitration showing time of appearance and rate of development ofcytopathic change is presented in FIG. 1. Both fourth and seventyseventhpassage level preparations contained 10 InD per 0.1 ml. when titrated inGMK cells with the use of the standard interference technique. As willbe seen from FIG. 1, virus in seventy-seventh passage (HPV77) firstproduced cytopathic changes at eight days and showed a titer of 10- per0.1 ml. by thirteen days. In contrast, cytopathic changes produced byfourth-passage virus occurred much later and progressed slowly. Evenafter twenty-five days of incubation the cytopathic end-point titer of10- TCD per 0.1 ml. was more than a bundred times less than theinfectivity titer obtained by measurement of interference (InD Table Ipresents further data concerning the comparative titration of severalpassage levels of the M-33 and ML strains in GMK and RK cell cultures.

TABLE I GMK cultures, 0 IIID 5e RKu cultures, Virus-passage level TOD eCell-culture passage level oi virus in African green monkey kidneycells.

b Logm tissue culture cytopathic (108950/1 .0 ml.

6 LOglO tissue culture interfering dose /1.0 ml.

6 Throat washing from ML.

Virus preparations were titrated by the usual methods.

Five to ten tube cultures of GMK or RK cells were each inoculated with0.1 ml. of serial tenfold dilutions made in Hanks BSS containing 1percent BPA. Titrations in GMK were tested for evidence of rubella-virusinterference with Echovirus Type 11 (E-11) after ten days; RK cells wereobserved for cytopathic effect (CPE) for twenty to twenty-one days.Fifty percent end points were calcuated by the Karber methods and wereexpressed as the interfering dose (InD or the tissue-culture cytopathic(105350 (TCDsu).

The relatively constant interference titers observed indicated noapparent change in behavior of the viruses as a result of continuedpassage. However, in RK cultures in which end points were based onappearance of CPE (TCD striking differences were noted betweenlowpassage and high-passage viruses. At GMK passage levels 2, 3, 4 and 9the onset and progression of CPE were delayed in a pattern similar tothat seen in FIG. 1, and the cytopathic end-point titers wereconsistently lower (1.3 to 4.0 log units) than the comparableinterference titration value. At higher passage levels (twenty-secondfor M-33 strain virus and twentieth for ML strain) this relation wasaltered so that titers indicated by the cytopathic system were alwaysequal to or higher than those shown by the interference method. Theperiod before onset of CPE was related to passage level; in 2experiments cytopathic changes in cultures inoculated withsixtieth-passage levels appeared and progressed to completion three tofive days earlier than twentieth or fortieth passages of the same virusstrains. Additional decreases in this period, as the number of passagesincreased from 60 to 100, were not apparent.

Thus, this technique provides a first signal of virus modification.

Plaque formation in RK ceIIs.The second of the laboratony markersutilized according to this invention is the formation of plaques in RKcells. Several GMK passage levels of strain M-33 and ML were titrated bythe interference technique and by plaque formation in RK cells. Acomparison of the values obtained is shown in the experiments summarizedin Table II.

e Celleulture passage level of virus in African green monkey kidneycells.

b Log plague-forming units (PFU)/0.2 ml. v Tissue-culture interfering(105850 (InDso)/0.2 ml. Throat washings from ML.

Rubella-virus plaque formation was assayed in RK cells; culturesreceived 0.2 ml. volumes of serial 0.5 log dilutions. After adsorptionfor one hour, inocula were removed, and the cultures overlayed with agarmedium.

Again, both strains behaved differently at low-passage and high-passagelevels. The M-33 strain in the fifth to the twenty-third passage and theML strain in either throat washing or fifth passage failed to produceplaques. Higher passage levels of both virus strains produced discrete,round plaques approximately 2 mm. in diameter after ten to fourteendays. In these experiments, 16 to 30 InD were required to produce 1 PFU.It is of interest that whereas twenty-third-passage M-33 virus producedmarked cyto- 'lfaylor-Robinson et al., "Plaque Formation by RubellaVirus, Lancet, vol. 1, p. 1364, 1964.

pathic change in RK cultures (Table I), plaque formation was notobserved.

This marker provides .yet another signal of virus modification.

Interferon pr0ductz'0n.--The third marker technique involved theproduction of interferon.

Assays of culture fluids of GMK cell cultures infected withfourth-passage and seventy-fourth-passage rubella virus rendered free ofinfectious virus by acidification to pH 1.0 were found to containinterferon. In preliminary experiments cultures infected with virus ofeither low or high passage produced interferon; however, the titersinduced by the high-passage virus were consistently greater than thoseby the low-passage virus. Results of a typical experiment are shown inTable III.

TAB LE III Interferon titer in indicated primary cel1-culture sysaCell-culture passage level of M-33 strain of rubella virus in GMK. bInterferon activity determined in homologous cell cultures with use ofvesicular-stomatitis-virus plague-assay method.

Interferon assays were performed in homologous cell cultures. Aftertwenty-four hours exposure to the interferon preparations cultures werechallenged with approximately 50 plaque-forming units ofvesicular-stomatitis virus (VSV). Plaques were counted after forty-eighthours, and the interferon titer Was expressed as the dilution resultingin a fifty percent reduction in the number of VSV plaques formed.

Interferon-like substances have been observed in infected primaryrabbit-kidney cell cultures. In the experiment shown, detectableinterferon was not induced by low-passage virus, butseventy-fourth-passage level induced a titer comparable to that observedin GMK. Other experiments in this system revealed that low-passage viruscould induce interferon, but the titers obtained were twofold tothreefold lower than those observed with highpassage material.

Thus, the in vitro marker techniques include (1) the production ofcytopathic effect in RK cells, (2) the formation of plaque in RK cells,and (3) the production of interferon titers in rabbit kidney cellscomparable to the titer in GMK. Each of these markers in and ofthemselves will not necessarily produce a clear indication of whichmaterial will prove useful as a vaccine, and which will not. However, byobserving the results of all three tests, those skilled in the art willbe provided with an indication of virus modification of the type Whichcan be expected to provide an antigenically active, attenuated, liverubella virus vaccine.

In order to support the accuracy of the in vitro marker techniques,evidence for modification of rubella viruses was also sought in in vivosystems. Earlier studies indicated that in rhesus monkeys inoculatedwith rubella virus a characteristic pattern of infection developed, thusproviding a useful experimental model. Several passage levels of theM-33 and ML virus strains were used to produce experimental infection inanimals inoculated intravenously or intramuscularly. Pharyngeal swab,rectal swab and blood specimens were collected at intervals of two orthree days for three weeks; serum specimens for antibody determinationswere obtained at intervals of two to eight days during the first monthafter inoculation and then bimonthly or monthly for as long as fourmonths. Monkeys were examined for signs of illness when specimens werecollected. None of the animals had clinical signs of rubella.

A comparison of the results of virologic studies on 24 monkeysinoculated parenterally with M-33 virus in thirdpassage to fifth-passagelevel and 14 given seventy-fourth passage are presented in Table IV.

V 12 The fact that the markers in Tables I and 11 showed an indicationof virus modification with twenty passage level ML strain virus is notcontradictory with the instant results. Although virus modification mayhave begun at TABLE IV Virus recovery from- Neutralizing- Transmissionto antibody response Pharynx Blood Rectum cage controls Number NumberNumber Number Number Number Number Number Number Number Virus-passagelevel positive tested positive tested positive tested positive testedpositive tested h Throat washings from ML.

Neutralizing antibody developed in all 24 animals inoculated withlow-passage virus and in 93 percent of those given high-passage virus.The pattern of virus recovery in the 2 groups was strikingly different.Of animals inoculated with third-passage to fifth-passage levels 50percent (12 of 24) exhibited viremia before the appearance of detectableantibody. Virus excretion from the respiratory tract was demonstrable in96 percent (23 of 24) of these animals on one or more occasions betweenthe fourth and seventeenth days after inoculation, and rectal excretionof virus was common. The inoculum in these monkeys ranged from 10 to 10InD of the low-passage virus. In contrast, no virus recoveries were madefrom animals inoculated with M-33 strain virus in seventy-fourthpassage. Of the 14 animals inoculated 9 received l0 to InDintravenously, and 5 were given 10 InD intramusc-ularly. These monkeysdid not show viremia or virus excretion from the respiratory orintestinal tract. In each experiment uninoculated monkeys (cagecontrols) were housed with those inoculated; other susceptible animalswere kept in adjacent cages (room controls). Serial serum specimens wereobtained from these control animals for at least eight weeks. Ingeneral, serologic evidence of contact infection with lowpassage viruswas observed in 13 to 67 percent of cage control animals; infection ofroom control monkeys was infrequent. In the experiments shown here,infection was transmitted to 2 of 3 cages mates and l of 4 roomcontrols. With high passage virus, experimental infections were nottransmissible. None of 14 cage controls had serologic evidence ofinfection. Moreover, rubella-virus antibody did not develop in 13 othermonkeys caged in the same room.

Limited data from studies with the ML strain are shown in the lowerportion of Table IV. The ML strain in throat washings, at levels offifth and twentieth GMK passage, was inoculated intravenously intogroups of 3 rhesus monkeys, each animal receiving from 10 to 10 InD ofvirus. Typical antibody responses developed in all. Virus was recoveredfrom blood and pharyngeal swabs ten to twenty days after inoculation.Each of the animals given throat washings from ML shed rubella virus.Either viremia or presence of virus in the pharynx was observed in all 3animals given fifth-passage virus, and in 2 of 3 inoculated withtwentieth-GMK-passage-level virus. Virus was not recovered from any ofthe specimens obtained from 1 animal in the last group. None of therectal-swab specimens collected from the 9 monkeys were positive.Infection was transmitted to the cake control of an animal inoculatedwith throat washings. Thus, serial passages of the ML strain in GMKcultures failed to produce the definite evidence of modificationapparent with seventyfourth passage-level M-33 strain rubella virusagain evidencing the need for high passage level to produce asatisfactory vaccine.

this lower level passage, attenuation had not yet been completed. Themarker techniques evidence this phenomenon by more subtle indicationsnot clearly brought out by the results shown in the tables. For example,as indicated above, in some experiments CPE in cultures inoculated withsixtieth passage level material appeared and progressed to completionsubstantially earlier than twentieth or fortieth level material of thesame strain. Similarly, interferon production increased with passagelevel indicating additional immunologic response at higher levels.

Other in vivo experiments in rhesus monkeys have been performed todetermine if the high-passage rubella virus produced pathologic changein the central nervous system. Seventy-seventh passage rubella virus wasinoculated directly into the central nervous systems of 30 monkeys. Tenwere inoculated with 0.5 ml. bilaterally into the thalamus and 0.25 ml.intracisternally. Twenty animals, pretreated with cortisone as in thestandard neurovirulence testing of attenuated poliovir us-vaccinestrains, were given 0.5 ml. bilaterally into the thalamus, 0.5 ml. intothe lumbar spinal cord and 1.0 ml. intramuscularly. The titer of thismaterial was 10 TCD per 0.5 ml. Animals were observed daily for clinicalsigns of neurologic disease. After twenty-one days the monkeys werekilled, and the brains and spinla cords removed by conventionaltechniques for pathological study. The findings paralleled thosepreviously reported for low-passage rubella-virus central-nervous systeminfections. There was no clinical evidence of neurologic disease.Sections of central-nervous system tissue, including lumbar and cervicalenlargements of' the spinal cord, medulla, pons, midbrain, cerebellumand cerebral hemispheres, failed to reveal any abnormality other thanthat resulting from the trauma of the inoculation procedure. Thus, noevidence for neuropathogenicity was found in monkeys inoculated witheither high-passage or low-passage levels of rubella virus.

Another important consideration in the production of a vaccine is theantibody response of animals inoculated with the same since this is anindication of an immunologic affect. The antibody response of animalsinoculated with seventy-fourth passage or seventy-seventh passage levelrubella virus resembled those that followed infection with throatwashings or third to twentieth tissue-culture passage preparations.Tests on serial serum samples from each group indicated that antibodywas first detectable between the ninth and twentieth days afterinoculation and increased to stable maximum titers by one or two months.Neutralizing-antibody titers ranged from 1:2 to 21:64, with a geometricmeans of 1:16. Regardless of virus-passage level, antibody titerpersisted essentially unchanged for at least three or four months afterinfection.

Antibody induced by previous rubella-virus infection has been shown toprotect monkeys against reinfection.

Table V presents data concerning the effect of rubella virus passagelevel or immunity to reinfection.

immunogenic and yet noncommunicable infections in vivo, were similar tothose of other successfully attenuated e Cell-culture passage level ofrubella virus in African green monkey kidney cells. Logio tissue-cultureinterfering (105950 of M-33 strain of rubella. virus inoculatedparentally.

Monkeys originally infected with high-passage and lowpassage levels ofvirus were challenged intravenously or intramuscularly several monthslater with fourth-passagelevel M-33 strain virus. Of 8 animalsoriginally infected with low-passage virus, 7 with antibody titers of1:4 to 1:64 showed no change in antibody level after challenge. In theremaining monkey with antibody detectable at a 1:2 serum dilution asixteenfold increase in titer developed. Virologic studies on 4 animalsimmunized four or five months previously with seventy-fourth-passagevirus and challenged intravenously with fourth-passage virus aresummarized in the lower portion of Table V. Two, selected because of lowpre-existing antibody levels (1:2 and 1:4), showed fourfold tosixteenfold increments in titer. The remaining animals, both with levelsof 1: 16 before challenge, did not show significant increases inantibody titer. Thus, challenge inoculation produced antibody increasesonly in animals with low levels of preexisting antibody. It is ofinterest that .despite parenteral challenge with to 10- lnD rubellavirus could not be recovered from animals with antibody resulting fromprior inoculation with high passage or low-passage rubella virus. Atotal of 68 pharyngeal, nasopharyngeal, rectal and blood specimenscollected after challenge from ani-' mals originally infected withlow-passage virus were negative. Virus-isolation specimens obtained fromthe 4 monkeys previously infected with high-passage virus did yieldvirus after challenge inoculation. None of these respiratory orintestinal tract swabs or heparipized 'blood specimens collected atintervals of two or three days during the three weeks after challengewere positive for rubella virus. There was no transmission of infectionfrom challenged animals to controls; none of 4 cage or 2 room controlmonkeys showed antibody.

The comparative studies performed in monkeys with low-passage andhigh-passage viruses provided confirmation of the attenuation indicatedby the in vitro laboratory marker techniques. The infection produced inthese animals by high-passage virus was drastically modified. Patternsof viremia, virus shedding and communicability characteristic oflow-passage virulent virus are not seen with high-passage virus. Thesechanges in experimental infections were not accompanied by developmentof neuropathogenicity. No evidence of pathologic changes incentral-nervous system tissues was found in rhesus monkeys inoculatedwith either lowor high-passages of rubella virus.

The observation that animals immunized with highpassage rubella viruswere protected against viremia and virus shedding by low-passagevirulent virus was of particular interest. This protective effect wasnot influenced by antibody level. Monkeys with low titers of antibodyhad antibody responses but, as in animals with higher titers, did notshed virus and were not viremic. It seems probable that animals withdetectable antibody would also be resistant to systemic infection underconditions of natural exposure. Since rubella-virus infections appear tobe transmitted to the products of conception via the maternalbloodstream absence of viremia would be expected to preclude fetalinfection and teratogenesis.

The alterations in the biologic characteriscs of highpassage rubellavirus, including increased cytopathic effect and interferon induction invitro and the production of viruses. Based primarily on the evidenceprovided by the marker techniques as confirmed by the animal tests,clinical trials of an experimental live-virus vaccine based on thehigh-passage virus (HPV-77) were initiated, although based on experiencewith other attenuated virus vaccines, similar experiences can beexpected at least with virus selected from passage levels 10 removed ineither direction from the tested 77th passage material. It is believedthat below about 67th passage level, the virus may not be sufficientlynoncommunicable, evidencing the importance of such high passage levelmaterial.

Production and testing of vaccine conformed to the rigorous requirementsapplying to commercially produced live measles and poliovirns vaccines.

Cultures of GMK cells grown in 32-02. ('846-ml.) bottles were inoculatedwith the high-passage M-33 strain. Before the period of virus harvestEagles minimal essential medium containing 1 percent fetal bovine serumand antibiotics was removed by washing of the monolayers three timeswith 50 ml. of Hanks BSS. Cultures were then re-fed and maintained onserum-free M-199 containing 25 g. per milliliter of neomycin as the onlyantibiotic. Daily harvests of infected fluids were made from the eighthto the fifteenth day after inoculation. Pooled fluids were centrifugedto remove intact cells. To the supernatant fluid commercially purchased,nonicterogenic, human serum albumin was added as a stabilizer to a finalconcentration of 2 percent. This seventy-seventh-GMK- passage material,after distribution in single dose containers, was frozen at 60 C. andrepresented the final vaccine.

When thawed and assayed in cell cultures the vaccine was found tocontain 10 TCD per 0.5 ml. of rubella virus. The vaccine has been stableunder these conditions of storage for in excess of six months.

Clinical studies with rubella-virus preparations free of detectableextraneous agents pose little risk if susceptible persons are shieldedfrom contact with those who have been vaccinated. Such carefullycontrolled conditions are best met in certain institutions. Here, thepopulation is more stable than in the community at large, and the entryof personnel into a study area is subject to administrative control.

After careful evaluation of all pertinent factors, an institution havingabout 700 students residing in widely scattered cottages that arefunctionally independent was selected. These arrangements made itpossible to isolate small groups of children from otherrubella-susceptible persons with minimal interruption in the normaleducational program. The immunity status of the 700 children and 300employees was ascertained in advance by virusneutralization tests.

Children were selected for participation in the study only after theirparents or legal guardians had been fully acquainted with all details ofthe project and had given written permission.

In various studies a total of 34 susceptible girls were inoculatedsubcutaneously with 0.5 ml. of experimental vaccine containingapproximately 10,000 TCD of the i live modified virus. The girls weremaintained in separate 15 cottage was isolated from other rubellasusceptible persons for from seven to eight weeks.

The children were examined daily, and their .temperatures recorded twicedaily for the seven or eight weeks duration of the study. The types ofspecimens collected for virologic testing included throat swabs dailyfor fiftytwo to fifty-seven days from both inoculated and uninoculatedparticipants, heparinized whole-blood samples on frequent occasionsbetween the sixth and twenty-first days from inoculated children andclotted blood for serologic examinations at weekly intervals for sevenor eight weeks from all members of the study groups. Throat swabs andheparinized blood samples were used for attempted virus recovery andthus were frozen immediately in Dry Ice.

Throat swabs, originally collected and frozen in 6.0 ml. of Hanks BSScontaining 1 percent BPA and antibiotics, were thawed and individuallyinoculated in 0.5 ml. volumes into each of 3 GMK culture tubes fromwhich the medium had been removed. After a one-hour adsorption period,the inoculum was replaced with 1.0 ml. of maintenance medium, and thetubes were incubated at 35 C. Tests for interfering viruses were madeafter ten days, both in the original inoculated cultures and in asubpassage. Interfering agents were identified as rubella virus byneutralization tests with specific immune serum.

Heparinized blood samples were inoculated similarly. After one hour theblood was removed, and cultures washed twice before refeeding. Again,tests for presence of virus were made by the interference technique.

To quantitate the amount of virus present, positive throat swab or bloodspecimens were titrated in tenfold dilutions in GMK and continuousrabbit kidney (RK tube cultures. Virus titers in GMK were expressed asthe interfering dose (InD and titers in RK as the cytopathic dose (TCDNeutralization tests were performed in RK tube cultures by methodspreviously described. Neutralizingantibody titers were expressed as thetwofold dilution of serum that protected 50 percent or more of culturesfrom rubella-virus cytopathic effect (CPE). Specimens that,

in the initial 1:2 dilution, failed to neautralize virus were considereddevoid of antibody.

Daily medical examinations for the test periods revealed no rash,vaccine-related fever or lymphadenopathy in any of the inoculatedchildren.

FIG. 2 summarizes the virologic events after HPV-77 virus inoculation ofthe 34 susceptible children.

Neutralizing antibody was first detected in 1 child on the twelfth day;most had antibody by the twenty-first day after inoculation. Numerousattemps were made to detect viremia. Although 7 to 18 specimenscollected daily from the sixth through the twenty-first days afterinoculation were tested rubella virus was not recovered from the bloodof any vaccinated child. Thus, none of 177 blood samples obtained fromthese children contained detectable virus.

The pattern of pharyngeal shedding of virus indicated that isolates maybe made from a few children as early as the seventh to the ninth day butare far more common between the tenth and twentieth days. The latestpositive throat swab was collected on the twenty-fourth day. Shedding ofvirus from the pharynx was frequently intermittent. Eleven of the 34inoculated never shed detectable virus, and even during the period ofmaximum virus excretion, no more than 34 percent of swab's were positiveon any given day. This pattern suggested that less Only one childexperienced any symptoms deserving special mention. this ten-year oldgirl streptococcal pharyng tis associated with rash developed. Theetiology of the infection was established by culture of Group A,fl-hemolytic streptococci and demonstration of a. rise inantistrepto1ysin-0 antibody titer. These finding and the occurence ofthe rash only eight days after inoculation led to the conclusion thatthe illness was not related to the vaccine.

- 16 virus is excreted in the attenuated virus infections that invirulent rubella.

There was no communicability evidenced during the clinical trials. Noneof the susceptible cottage contacts of the vaccinated children developedantibody or shed virus during the observation period.

The level and duration of neutralizing antibody are depicted in FIG. 3wherein the circled figures represent the number of serum samplesnegative for antibody on the days specified. In 32 of 34 children, or 94percent, neutralizing antibody developed by the twenty-eighth day afterinoculation. Antibody titers averaged 1:8 and ranged from 1:2 to 1:64,four weeks after infection.

By chance it was possible to study a virulent outbreak of rubella-virusinfection at the same institution, the outbreak being confined to asingle cottage not involved in the vaccine study. The natural diseasewas studied with the same virologic methods used to evaluate thevaccineinduced infections.

One of the girls from this other cottage was exposed to a child with arash outside of the institution and on her return, seventeen days afterexposure, rubella developed. Ten other rubella-susceptible chlidren werein the cottage; in the ensuing eight weeks all 10 were infected by thevirulent rubella virus. There was no spread of the virus to personsoutside the cottage.

The clinical features of natural rubella as compared to the infectionsproduced by the attenuated virus are listed in Table VI.

TABLE VI Number of children- With Wlth tnapparent Type of virus incottage Infected rash infections Of the 11 persons infected withvirulent rubella during the outbreak, 9 experienced the typical diseasewith rash, and 2 had clinically inapp'arent infections. In contrast,none of the 34 rubella-susceptible children living in the test cottageshad any symptoms of rubella after inoculation with high-passage virus. I

The results of virus studies in the cases of natural rubella aresummarized in FIG. 4. There was a high efiiciency of virus recovery fromthroat-swab specimens. During the period from seven days before theonset of rash to four days afterward, 26 of 28 throat swabs collected in8 of the secondary cases with clinical rubella yielded virus. Forseveral consecutive days percent of swabs were positive. To demonstrateviremia, it is desirable to obtain blood samples during the periodbefore the onset of rash. Of the 4 heparinized blood samples collectedone to four days before the onset of rash 3 were definitely positive forrubella virus. This is atypical virologic picture of virulent rubellaand stands in contrast to the experience with vaccinated children. Inthe attenauted-virus infection (FIG. 2) viremia was never shown, and onany given day not more than 34 percent of throat swabs were positive.

The outbreak of virulent rubella provided an opportunity to compare thecommunicability of natural and attenuated rubella infections in theparticular environmental setting as shown in Table VII.

TABLE VII Number oi Suscep- Type of virus in tible Contacts cottagecontacts infected Orlginal exposure to one patient with virulent li l ae h group exposed to 8 to 10 children infected with HPV-77 rubellavirus.

In the cottage having virulent virus, all of 10 susceptible personsexposed to the natural rubella were infected during the following eightweeks. In each of the other cottages, 6 to 10 vaccinated childreninfected with the attenuated virus lived with 7 or 8 susceptibleplaymates. None of these 30 intimate contacts were infected.

Because of the obvious differences in the communicability of thevirulent and attenuated-virus infections, attempts were made toquantitate the amount of virus excreted. Thirteen virus-positivethroat-swab specimens from the virulent-rubella outbreak and 22 positivethroat swabs from vaccinated children infected with the attenuated viruswere titrated in tissue cultures. The geometric mean titer of virus inswabs from the virulent cases was 10- per 0.1 ml., and that from thevaccinated children was only per 0.1 ml., representing a hundred-folddifferencethat is, 320 as compared to 2.5 infectious doses.

The laboratory marker tests that had indicated modification of the highpassage virus were used to characterize the viruses excreted by thevaccinated children as will be seen below in Table VIIII.

TABLE VIII Rubella-virus type A representative isolate from aninoculated girl was subjected to the entire gamut of tests. Each of themarker tests indicated that the isolate possessed the properties of thevaccine strain and not of virulent rubella virus. The isolate strainproduced cytopathic changes (CPE) and plaques in RK cultures and inducedthe production of interferon in vitro. Interferon titers in GMK culturesinfected with the isolate were 1:13 as compared to less than 1:4 withlow-passage virus. Five rhesus monkeys inoculated intravenously with thevirus from the vaccinated girl were not viremic, did not shed virus anddid not transmit their infections to uninoculated cagemates.

With the use of the RK cytopathology marker 22 other representativepharyngeal isolates from 8 vaccinated children were compared with 16isolates from the pharynx and blood of 4 children involved in theoutbreak of virulent rubella. None of the viruses from the cases ofnatural rubella produced CPE in RK cultures whereas all the strains fromthe vaccinated children did. Thus, the viruses recovered from vaccinatedchildren uniformly exhibited the properties characteristic of thehigh-passage attenuated strain.

Continuing these investigations of qualitative differences, experimentswere done to compare the infectivity of low-passage and high-passagerubella viruses in rhesus monkeys inoculated by the intranasal route.Tenfold dilutions of each virus were inoculated intranasally into groupsof 5 susceptible monkeys. Infectivity end points of the 2 viruspreparations were calculated on the basis of appearance of antibody inthe animal as seen in Table IX.

B Virus titer/ml.

Assay of the pools of virulent and attenuated virus in tissue culturesshowed titers of l0- and 10 TCD per 1.0 ml., respectively. Afterintranasal inoculation into monkeys the infectivity end points were 1()and 10', respectively, indicating that the attenuated strain was farless eflicient than the virulent in inducing simian infection by theintranasal route.

Thus, it will now be seen that there is herein provided methods for theproduction of an attenuated, immunogenic, noncommunicable live virusrubella vaccine and laboratory techniques for indicating modification ofthe virulent virus. The general pattern of neutralizing-antibodyresponse to infection with the high-passage virus of this inventionresembles that observed in natural rubella. Many studies haveestablished that the presence of rubella neutralizing antibody preventsboth clinical and subclinical infections in man and experimentallyinoculated monkeys. Thus, it seems likely that children infected withthe attenuated virus will be immune to the disease. The absence ofcommunicability in studies with 34 vaccinated and 30 control children in4 different cottages is significant. In general, institutionalizedchildren experience high attack rates with communicable disease as areflection of conditions of intimate contact. The natural rubellaoutbreak in which percent of susceptible children were infecteddemonstrates the high secondary attack rate of rubella-virus infectionsunder these circumstances. v

The quantitative difference in the virus-excretion patterns of virulentand attenuated rubella infections may well be important in explainingthe lack of communicability observed with the latter. On the basis oftissue-culture assay data it appears that about a hundred times morevirus is shed in natural rubella. Even if infection were transmittedfrom a vaccinated child to an uninoculated person there seems to be lessrisk that further spread would result. The results of laboratory markertests indicated that the properties of the viruses excreted by thevaccinated subjects were the same as those of the attenuated strain. Itseems likely that a secondary infection, it it occurred, would also beattenuated and relatively noncommunicable.

The qualitative differences in the attenuated and virulent viruses shownby the intranasal inoculation of monkeys extend the theoretical marginof safety.

Since there are modifications of the instant inventive concepts whichwill be obvious to those skilled in the art, all matter herein is to beconsidered illustrative and no in a limiting sense unless otherwiseidentified.

What is claimed is:

1. A method for preparing an attenuated live rubella virus comprisingthe steps of introducing a virulent live rubella virus into a monkeykidney tissue cell culture, incubating said tissue culture at atemperature compatible with growth of said tissue and said virus,harvesting at least a portion of the virus so-produced and reintroducingsaid harvested virus into fresh cultures of the selected tissue, andrepeating such tissue culture passages of the virus serially for asufficient number of passages to produce an antigenically active,noncommunicable, live rubella virus.

2. The method of claim 1 wherein said tissue culture passages arerepeated serially through at least about 67 passages, and including thestep of forming a vaccine from such high passage virus.

3. The method of claim 2 wherein said virus is of the M-33 rubellastrain and said tissue culture passages are repeated serially throughabout 77 passages.

4. A method for preparing an attenuated live rubella virus vaccinecomprising the steps of introducing a virulent live rubella virus into aprimary African green monkey kidney cell culture, incubating said tissueculture at about 35 C., harvesting at least a portion of the virussoproduced at approximately 7 to 19 day intervals and reintroducing saidharvested virus into fresh primary African green monkey kidney cellcultures, repeating such tissue culture passages of the virus seriallyfor between about 67 and 87 passages, and concentrating such highpassage virus to form a vaccine therefrom.

5. The method of claim 4 whereas said rubella virus is of the M-33strain and said tissue culture passages are repeated serially for about77 passages.

6. The method of claim 4 wherein said rubella virus is of the ML strain.

7. A process for producing an attenuated live rubella virus whichcomprises the steps of introducing a virulent live rubella virus into amonkey kidney tissue cell culture, incubating said tissue culture at atemperature compatible with the growth of said tissue and said virus,harvesting at least a portion of the virus so produced, reintroducingsaid harvested virus into fresh culture of the tissue, and repeatingsuch tissue culture passage for a sufficient number of pasages toproduce an antigenically-active, noncommunicable, live rubella virus.

8. An attenuated live rubella virus vaccine containing an eifectivequantity of an antigenically-active, non-communicable, live rubellavirus produced by introducing a virulent live rubella virus into amonkey kidney cell culture incubating said tissue culture at about C.,harvesting at least a portion of the virus so prodced at approximately 7to 10 day intervals and reintroducing said harbested virus into freshcell cultures of the same type, repeating such tissue culture pasages ofthe virus serially for at least passages, and concentrating theresulting viruses to form a vaccine therefrom.

References Cited UNITED STATES PATENTS 3,401,084 9/1968 Buynak et a1.195-l.3

RICHARD L. HUFF, Primary Examiner US. Cl. X.R. ll.3

